We are interested in the mechanistic and molecular relationships between catalytic activity, conformational changes and microenvironment of ABC transporters. P-glycoprotein (ABCB1, Pgp) is in the focus of our interest; we have currently extended our work to ABCG2 (BCRP) and plan to do similar studies on MRP1 (ABCC1). The members of the ABC superfamily of membrane transporters are involved in the regulation of the uptake into and distribution within our body of physiological substrates as well as various xenobiotics, drugs. Due to their wide substrate spectrum, a consequence of their preference for lipophylic compounds, they also play a critical role in the multidrug resistance phenomenon severely limiting therapeutical success in cancer. Our ambition is to understand the molecular details of their catalytic cycle and the intimate molecular interactions with their microenvironment, as well as to apply the knowledge obtained at the cell/molecule level in the context of the whole organism, in vivo.

Our experimental approaches include: (a) sensitive flow-cytometric methods to detect substrate efflux; (b) ATPase assays to measure the interaction of drugs with ABC transporters in membrane fractions; (c) intracellular ATP concentration measurements in live cells using a FRET-based intracellular ATP sensor; (d) a flow-cytometric platform for the measurement of raft- and cytoskeleton-association of cell-surface proteins, applicable also for rapid, simple, serial analysis of intermolecular associations between different cell surface proteins; (e) immunoprecipitation and co-immunprecipitation to study protein-protein interactions by Western-blot and MALDI analysis; (f) a sensitive flow cytometric assay to measure ATP dependent conformational changes of Pgp and ABCG2 in Staphylococcus toxin permeabilized cells; (g) a confocal microscopic assay to measure substrate binding to transporters in live as well as permeabilized cells; (h) xenotransplantation system in SCID mice to study the effect of ABC transporter modulators on multidrug resistant tumors by PET.

Due to the repressive nature of nucleosomes, the main underlying scheme of gene regulation in eukaryotic cells is de-repression. On the other hand, nucleosome stability is highly sensitive to the degree of superhelicity, a transcription dependent feature of the matrix anchored DNA loops. However, at their anchorage points, genomic DNA is relaxed due to strand discontinuities arising in conjunction with transcription, likely introduced by topoisomerase enzymes. We are investigating the molecular determinants and dynamic features of these two distinct chromatin domains in the context of gene activition. Our ambition is to unravel the complex relationships between nucleosome stability and organization of superhelical chromatin loops, and understand the relationships between the organizational principles seen at the loop level and those of global nuclear compartmentalization.